1. Field of the Invention
The present invention relates, in general, to a DNA segment. In particular, the present invention relates to a DNA segment comprising a selectable marker gene, a DNA segment comprising a selectable marker gene inserted into a retrotransposon, cells containing these DNA segments, and methods of using these DNA segments.
2. Background Information
Ty elements of Saccharomyces cerevisae are retrotransposons that are similar to retroviral proviruses (Boeke, J.D. (1989) in Mobile DNA, eds., Berg, D. E. & Howe, M. M. (Am. Soc. Microbiol, Washington), pp. 335-374). Retrotransposition is a replicative process involving reverse transcription of Ty mRNA and integration of Ty cDNA into the genome (Boeke et al. Cell (1985) 40: 491-500). Ty1 elements are the most common insertional mutagen and comprise the most numerous family of the four Ty element classes, with about 25-30 copies of Ty1 per haploid genome (Cameron, et al. Cell (1979) 16:739-751; Curcio, M. J. et al. Mol. Gen. Genet.(1990) 220:213-221). Despite the fact that Ty1 RNA accounts for 1% of total yeast RNA (Curcio, M. J. et al. Mol. Gen. Genet. (1990) 220:213-221), the rate of transposition is quite low (Giroux, C. N., et al. Mol. Cell. Biol. (1988) 8:978-981; Boeke, J. D. et al. Mol. Cell. Biol. (1986) 6:3575-3581; Paquin, C. E. et al. Mol. Cell. Biol. (1986) 4:70-79). Several modulators of transposition have been described. For example, Ty transposition is stimulated at temperatures below 30.degree. C. (Paquin, C. E., et al. Science (1984) 226:53-55), by exposure of the cells to ultraviolet irradiation or 4-nitroquinoline 1-oxide (Bradshaw, V. A., et al. Mol. Gen. Genet. (1988) 218:465-474), or in a rad6 mutant background (Picologlou, et al. Mol. Cell. Biol. (1990) 10: 1017-1022). Mutations in the SPT3 gene alter the initiation of Ty1 transcription (Winston, et al. Cell (1984) 39:675-682) and abolish retrotransposition of chromosomal Ty1 elements (Boeke, J. D. et al. Mol. Cell. Biol. (1986) 6:3575-3581). These modulators of retrotransposition were identified by their effect on the frequency of Ty insertions into specific loci and not into the genome as a whole. As a result, it can be difficult to determine whether the modulators alter Ty elements directly or the target locus (Picologlou, et al. Mol. Cell. Biol. (1990) 10: 1017-1022).
A tremendous induction in the rate of Ty1 transposition is achieved by expressing an active Ty element, Ty1-H3, from the inducible GAL1 promoter (Boeke et al. Cell (1985) 40: 491-500). The pGTy1-H3 element has been marked with selectable genes such as a bacterial gene for neomycin resistance (Boeke, et al. Science (1988) 239:280-282) and the yeast HIS3 gene (Garfinkel, et al. Genetics (1988) 120:95-108). Phenotypic detection of retrotransposition events in the transposition-induction system requires loss of the pGTy plasmid. In addition, transposition of the marked Ty1 element can only be detected when it is induced to a level that exceeds the rate of homologous recombination among Ty elements (Roeder, et al. Proc. Natl. Acad. Sci. USA (1982) 79:5621-5625; Roeder, et al. Mol. Cell. Biol. (1984) 4:703-711).